Distortion of the L→M transition in the photocycle of the bacteriorhodopsin mutant D96N: a time-resolved step-scan FTIR investigation

Abstract

The D96N mutant of bacteriorhodopsin has often been taken as a model system to study the M intermediate of the wild type photocycle due to the long life time of the corresponding intermediate of the mutant. Using time-resolved step-scan FTIR spectroscopy in combination with a sample changing wheel we investigated the photocycle of the mutant with microsecond time resolution. Already after several microseconds an intermediate similar to the M N state is observed, which contrasts with the M state of the wild type protein. At reduced hydration M and N intermediates similar to those of wild type BR can be detected. These results have a bearing on the interpretation of the photocycle of this mutant. A mechanism is suggested for the fast rise of M N which provides some insight into the molecular events involved in triggering the opening of the cytosolic channel also of the wild type protein.